The invention relates to the formation of peptide/lipid vesicles and complexes through the co-lyophilization of peptides, preferably that are able to adopt an amphipathic alpha-helical conformation, and one or more lipids. A single solution which solubilizes both the peptides and lipids or a two separate solutions may be lyophilized. The methods are used to generate stable peptide/lipid vesicles and complexes including but not limited to micellar, spherical and discoidal complexes in bulk preparations and in smaller units, as may be suitable for dosages.
Liposomes are vesicles composed of at least one lipid bilayer membrane enclosing an aqueous core. Generally, phospholipids comprise the lipid bilayer, but the bilayer may be composed of other lipids. The aqueous solution within the liposome is referred to as the xe2x80x9ccaptured volume.xe2x80x9d Liposomes have been developed as vehicles to deliver drugs, cosmetics, bioactive compounds among other applications. The lipid bilayer encapsulates the drug, cosmetic, bioactive compound, and the like, within the captured volume of the liposome and the drug is expelled from the liposome core when the lipid bilayer comes in contact with a cell surface membrane. The liposome releases its contents to the cell by lipid exchange, fusion, endocytosis, or adsorption. Ostro et al., 1989, Am. J. Hosp. Pharm. 46:1576. Alternatively, the drug, cosmetic, bioactive compound and the like could be associated with or inserted into the lipid bilayer membrane of the vesicle.
In addition to vesicles, lipid-containing complexes have been used to deliver agents in particle form. For instance, many researchers have found it useful to prepare reconstituted lipoprotein-like particles or complexes which have similar size and density as high density lipoprotein (HDL) particles. These reconstituted complexes usually consist of purified apoproteins (usually apoprotein A-1) and phospholipids such as phosphatidylcholine. Sometimes unesterified cholesterol is included as well. The most common methods of preparing these particles are (1) co-sonication of the constituents, either by bath sonication or with a probe sonicator, (2) spontaneous interaction of the protein constituent with preformed lipid vesicles, (3) detergent-mediated reconstitution followed by removal of the detergent by dialysis. Jonas, 1986, Meth. in Enzymol. 128:553-582; Lins et al., 1993, Biochimica et Biophysica Acta, 1151:137-142; Brouillette and Anantharamaiah, 1995, Biochimica et Biophysica Acta, 1256:103-129; Jonas, 1992, Structure and Function of Apoproteins, Chapter 8:217-250. Similar complexes have also been formed by substituting amphipathic helix-forming peptides for the apoprotein components. Unfortunately, each of these methods presents serious problems for the formation of large amounts of pure complexes on a reasonably cost-effective basis. Further, none of these publications disclose the co-lyophilization of peptides/or peptides analogues which are able to adopt an amphipathic alpha helical conformation and a lipid.
A range of technologies is known for producing lipid vesicles and complexes. Vesicles, or liposomes, have been produced using a variety of protocols, forming different types of vesicles. The various types of liposomes include: multilamellar vesicles, small unilamellar vesicles, and large unilamellar vesicles.
Hydration of phospholipids (or other lipids) by aqueous solution can also result in the dispersion of lipids and spontaneous formation of multimellar vesicles (xe2x80x9cMLVsxe2x80x9d). An MLV is a liposome with multiple lipid bilayers surrounding the central aqueous core. These types of liposomes are larger than small unilamellar vesicles (SUVS) and may be 350-400 nm in diameter. MLVs were originally prepared by solubilizing lipids in chloroform in a round-bottom flask and evaporating the chloroform until the lipid formed a thin layer on the wall of the flask. The aqueous solution was added and the lipid layer was allowed to rehydrate. Vesicles formed as the flask is swirled or vortexed. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citing Bangham et al., 1965, J. Mol. Biol. 13:238). Johnson et al. subsequently reported that this method also generated single lamellar vesicles. Johnson et al., 1971, Biochim. Biophys. Acta 233:820.
A small unilamellar vesicle (SUV) is a liposome with a single lipid bilayer enclosing an aqueous core. Depending on the method employed to generate the SUVS, they may range in size from 25-110 nm in diameter. The first SUVs were prepared by drying a phospholipid preparation in chloroform under nitrogen, adding the aqueous layer to produce a lipid concentration in the millimolar range, and sonicating the solution at 45xc2x0 C. to clarity. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. SUVs prepared in this fashion yielded liposomes in the range of 25-50 nm in diameter.
Another method of making SUVs is rapidly injecting an ethanol/lipid solution into the aqueous solution to be encapsulated. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citing Batzri et al., 1973, Biochim. Biophys. Acta 298:1015). SUVs produced by this method range in size from 30-110 nm in diameter.
SUVs may also be produced by passing multilamellar vesicles through a French Press four times at 20,000 psi. The SUVs produced will range in size from 30-50 nm in diameter. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citing Barenholz et al., 1979, FEBS Letters 99:210).
Multilamellar and unilamellar phospholipid vesicles can also be formed by extrusion of aqueous preparations of phospholipids at high pressure through small-pore membranes (Hope et al., 1996, Chemistry and Physics of Lipids, 40:89-107)
A large unilamellar vesicle (LUV) is similar to SUVs in that they are single lipid bilayers surrounding the central aqueous core, but LUVs are much larger that SUVs. Depending on their constituent parts and the method used to prepare them, LUVs may range in size from 50-1000 nm in diameter. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. LUVs are usually prepared using one of three methods: detergent dilution, reverse-phase evaporation, and infusion.
In the detergent dilution technique, detergent solutions such as cholate, deoxycholate, octyl glucoside, heptyl glucoside and Triton X-100 are used to form micelles from the lipid preparation. The solution is then dialyzed to remove the detergent and results in the formation of liposomes. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York. This method is time consuming and removal of the detergent is generally incomplete. The presence of detergent in the final preparation may result in some toxicity of the liposome preparation and/or modification of the physicochemical properties of the liposome preparation.
The reverse-phase evaporation technique solubilizes lipid in aqueous-nonpolar solutions, forming inverted micelles. The nonpolar solvent is evaporated and the micelles aggregate to form LUVs. This method generally requires a great deal of lipid.
The infusion method injects a lipid solubilized in a non-polar solution into the aqueous solution to be encapsulated. As the nonpolar solution evaporates, lipids collect on the gas/aqueous interface. The lipid sheets form LUVs and oligolamellar liposomes as the gas bubbles through the aqueous solution. Liposomes are sized by filtration. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York (citing Deamer et al., 1976, Biochim. Biophys. Acta 443:629 and Schieren et al., 1978, Biochim. Biophys. Acta 542:137). Infusion procedures require a fairly high temperature for infusion and may have a relatively low encapsulation efficiency. Deamer et al., 1983, in Liposomes (Ostro, Ed.), Marcel Dekker, Inc. New York
It is has been a goal of liposome research to develop liposome preparations that may be stored for long periods of time before use. For example, U.S. Pat. No. 4,229,360 to Schneider et al., discloses a method of dehydrating liposomes by adding a hydrophilic compound to a colloidal dispersion of liposomes in an aqueous liquid and dehydrating the solution, preferably by lyophilization. Examples of hydrophilic compounds are high molecular weight hydrophilic polymers or low molecular weight compounds such as sucrose.
U.S. Pat. No. 4,411,894 to Shrank et al., discloses the use of high concentrations of sucrose in sonicated preparations of liposomes. The liposomes contain fat-soluble products in the captured volume, although the preparations could be lyophilized, the method could not prevent the loss of a significant amount of the captured contents despite the high concentration of sucrose.
Crowe et al., U.S. Pat. No. 4,857,319 disclosed the use of disaccharides such as sucrose, maltose, lactose and trehalose to stabilize liposomes when liposomes are freeze dried. The amount of disaccharide with respect to the lipid content of the component (w/w) is within 0.1:1 to 4:1. Crowe achieved greater success in preserving liposomal integrity using this method than that afforded by the method disclosed by Shrank in U.S. Pat. No. 4,441,894.
Janoff et al, U.S. Pat. No 4,880,635 disclose a method for dehydrating liposomes in which liposomes were lyophilized in the presence of protective sugars such as trehalose and sucrose, preferably on both the inner and outer leaflets of the lipid bilayer. Sufficient water is retained in the method of Janoff et al. so that rehydration of the dried liposomes yields liposomes with substantial structural integrity.
However, there is a need in the art for a simple and cost effective method of forming lyophilized peptide/lipid complexes which may be then be rehydrated. The method of the present invention yields peptide/lipid mixtures in a stable, lyophilized powder which may be stored, used as a powder, or used after rehydration to form peptide/lipid complexes.
The invention is a method for preparing peptide or protein-(phospho)lipid complexes or vesicles which may have characteristics similar to high density lipoprotein (HDL). The method utilizes a solvent system in which at least one peptide is solubilized in one solution, and at least one lipid is solubilized in another solution. The two solutions are selected such that they are miscible with one another. The solutions are then combined, and the resulting solution is lyophilized.
The method also may be practiced by a second type of solvent system comprising a solution into which both the protein or peptide and the lipid may be solubilized. This solution may be a single solution, or may be a composite solution made by combining two or more solutions before the addition of peptides and lipids. Peptides and lipids are solubilized in the solution or composite solution and the peptide/lipid solution is then lyophilized.
Preferably, the peptides of the present invention are peptides which are able to adopt an amphipathic helical conformation. In one specific embodiment of the invention, the peptide is a lipid binding protein. In other embodiment, peptide analogues of ApoA-I, ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE, other apolipoprotein analogues and the like are utilized in place of or in combination with the peptides. In another specific embodiment, the method is used to prepare ApoA1 analogue/(phospho)lipid complexes similar to HDL. The ApoA1/lipid complexes are useful in treating disorders associated with dyslipoproteinemias including but not limited to hypercholesterolemia, hypertriglyceridemia, low HDL, and apolipoprotein A-1 deficiency, septic shock, for in vitro diagnostic assays as markers for HDL populations, and for use with imaging technology.
The method of the invention enables the preparation of peptide/lipid complexes for parenteral administration including but not limited to intravenous, intraperitoneal, subcutaneous, intramuscular, and bolus injections to animals or humans. Further, the peptide/lipid complexes can also be formulated for oral, rectal, mucosal (e.g. oral cavity) or topical administration to animals or humans, or for in vitro experimentation.
The method may be used for large scale production of amphipathic peptide/phospholipid complexes, lipid binding protein/phospholipid complexes, and/or ApoA1 peptide analogue/phospholipid complexes. The lyophilized material may be prepared for bulk preparations, or alternatively, the mixed peptide/lipid solution may be apportioned in smaller containers (for example, single dose units) prior to lyophilization, and such smaller units may be prepared as sterile unit dosage forms.
The lyophilized powder prepared by the method of the invention can be rehydrated into a particulate-free sterile solution immediately before injection, or alternatively, the lyophilized powder can be formulated into an appropriate solid dosage form and administered directly.
The method may also be suitable for storage of compounds which may be otherwise unstable or insoluble in the absence of lipids.
The method may be used for the formulation of products for the treatment or prevention of human diseases, including such applications as co-presentation of antigens in vaccines, treatment or prevention of dyslipoproteinemias, including but not limited to hypercholesterolemia, hypertriglyercidemia, low HDL, and apolipoprotein A-1 deficiency, cardiovascular disease such as atherosclerosis, septic shock, or infectious diseases.
The method may be used for the preparation of complexes that could be used as carriers for drugs, as vectors (to deliver drugs, DNA, genes), for example, to the liver or to extrahepatic cells, or as scavengers to trap toxin (e.g. pesticides, LPS, etc.).
As used herein, a xe2x80x9csolvent systemxe2x80x9d refers to one or more solvents which are capable of solubilizing peptides and/or lipids and, if more than one, which are miscible with one another.
As used herein, xe2x80x9cpeptide/lipid complexesxe2x80x9d refers to an aggregation of lipid moieties and peptides forming particles within the size range of high density lipoproteins (HDLs).
As used herein, xe2x80x9cco-lyophilizedxe2x80x9d refers to the lyophilization, freeze-drying, or vacuum drying of more than one compound (e.g., peptide, protein, lipid, phospholipid) in solution in the same vessel. For example, a lipid solution may be combined with a peptide solution in the same vessel and the resulting combination of solutions is lyophilized together, thereby lyophilizing the peptides and lipids simultaneously.
As used herein xe2x80x9camphipathic peptidexe2x80x9d or xe2x80x9camphipathic alpha helical peptidesxe2x80x9d means peptides which are able to adopt an amphipathic or amphipathic helical conformation, respectively. The amphipathic alpha helix is an often encountered secondary structural motif in biologically active peptides and proteins. See AmphiPathic helix motif: classes and properties by Jere P. Segrest, Hans de Loof, Jan G. Dohlman, Christie G. Brouillette, and G. M. Anantharamaiah. PROTEINS: Structure Functions and Genetics 8:103-117 (1990). An amphipathic alpha helix is an alpha helix with opposing polar and nonpolar faces oriented along the long axis of the helix. A specific distribution of charged residues is evident along the polar face. Amphipathic helices, as defined, are complementary for the polar-nonpolar interface of hydrated bulk phospholipid; these lipid-associating domains have been postulated to interact with the phospholipid by partially immersing themselves at the interface between the fatty acyl chains and the polar head groups. Jere P. Segrest. Febs letters 1976, 69 (1): 111-114.
The term xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d may be used interchangeably herein. Further, the peptide analogues of the invention can be peptides, proteins or non-peptides i.e., peptidomimetics. However, all the analogues are preferably bioactive molecules.
The term xe2x80x9clipidxe2x80x9d as used herein includes but is not limited to natural and synthetic phospholipids. Further, the terms, xe2x80x9cLipidxe2x80x9d and xe2x80x9cphospholipidxe2x80x9d may be used interchangeably herein.
FIG. 1: Superose 6 chromatography of HDL prepared by density ultracentrifugation from 200 xcexcl human serum.
FIG. 2 (bottom): Superose 6 chromatography of (DPPC: peptide 1) (PVLDLFRELLNELLEALKQKLK; SEQ ID NO:1) complexes prepared at a ratio of 1:1 (w:w).
FIG. 2 (top): Superose 6 chromatography of (DPPC: peptide 1) complexes prepared at a ratio of 2:1 (w:w).
FIG. 3 (bottom): Superose 6 chromatography of (DPPC: peptide 1) complexes prepared at a ratio of 3:1 (w:w).
FIG. 3 (top): Superose 6 chromatography of (DPPC: peptide 1) complexes prepared at a ratio of 4:1 (w:w).
FIG. 4 (bottom): Superose 6 chromatography of (DPPC: peptide 1) complexes prepared at a ratio of 5:1 (w:w).
FIG. 4 (top): Superose 6 chromatography of (DPPC: peptide 1) complexes prepared at a ratio of 7.5:1 (w:w).
FIG. 5: Superose 6 chromatography of (DPPC: peptide 1) complexes prepared at a ratio of 10:1 (w:w).
FIG. 6: Superose 6 chromatography of 14C-labeled peptide 1 complexes at Ri=3:1.
FIG. 7: Superose 6 chromatography of 14C-labeled peptide 1 complexes at Ri=4:1.
FIG. 8: Superose 6 chromatography of 14C-labeled peptide 1 complexes at Ri=5:1.